A kindred of multiple endocrine neoplasia type 2B.

We describe familial cases of multiple endocrine neoplasia (MEN) 2B: A 48-year-old man is the proband. He had pheochromocytoma, medullary thyroid carcinomas (MTCs), parathyroid hyperplasia, mucosal neuromas, eversion of eyelids and Marfanoid appearance, and then underwent adrenalectomy and total thyroidectomy. Family screening revealed that his two daughters (10 and 8 years old) had mucosal neuromas and increased serum calcitonin (CT). Both of them had MTCs but no pheochromocytoma, and their MTCs were surgically removed. The father and his children have been in favorable condition since the operations. Southern blot analysis with 33 polymorphic DNA probes was done in MTCs obtained from two daughters. An RBP3 (10q11.2) locus linked to a predisposing gene on chromosome 10 was uninformative in either patient because of constitutional homozygosity. Loss of heterozygosity at the MYCL1 locus on chromosome 1p32 was observed in MTC from the younger sister, but no loss of heterozygosity was recognized in other loci examined. Deletion of the 1p32 locus may play a role in the development of MEN 2B.     

A Drosophila model of multiple endocrine neoplasia type 2

Dominant mutations in the Ret receptor tyrosine kinase lead to the familial cancer syndrome multiple endocrine neoplasia type 2 (MEN2). Mammalian tissue culture studies suggest that RetMEN2 mutations significantly alter Ret-signaling properties, but the precise mechanisms by which RetMEN2 promotes tumorigenesis remain poorly understood. To determine the signal transduction pathways required for RetMEN2 activity, we analyzed analogous mutations in the Drosophila Ret ortholog dRet. Overexpressed dRetMEN2 isoforms targeted to the developing retina led to aberrant cell proliferation, inappropriate cell fate specification, and excessive Ras pathway activation. Genetic analysis indicated that dRetMEN2 acts through the Ras-ERK, Src, and Jun kinase pathways. A genetic screen for mutations that dominantly suppress or enhance dRetMEN2 phenotypes identified new genes that are required for the phenotypic outcomes of dRetMEN2 activity. Finally, we identified human orthologs for many of these genes and examined their status in human tumors. Two of these loci showed loss of heterozygosity (LOH) within both sporadic and MEN2-associated pheochromocytomas, suggesting that they may contribute to Ret-dependent oncogenesis.     

Parent-of-origin effects in multiple endocrine neoplasia type 2B.

Multiple endocrine neoplasia type 2B (MEN 2B) is characterized by medullary thyroid carcinoma, pheochromocytomas, mucosal neuromas, ganglioneuromas, and skeletal and ophthalmic abnormalities. It is observed as both inherited and sporadic disease, with an estimated 50% of cases arising de novo. A single point mutation in the catalytic core region of the receptor tyrosine kinase, RET, has been observed in germ-line DNA of MEN 2B patients. We have analyzed 25 cases of de novo disease in order to determine the parental origin of the mutated RET allele. In all cases the new mutation was of paternal origin. We observe a distortion of the sex ratio in both de novo MEN 2B patients and the affected offspring of MEN 2B transmitting males. These results suggests a differential susceptibility of RET to mutation in paternally and maternally derived DNA and a possible role for imprinting of RET during development.     

Familial medullary thyroid carcinoma and multiple endocrine neoplasia type 2B map to the same region of chromosome 10 as multiple endocrine neoplasia type 2A.

Medullary thyroid carcinoma (MTC) occurs as a component of three well-described autosomal dominant familial cancer syndromes. Multiple endocrine neoplasia type 2A (MEN 2A) is characterized by MTC, pheochromocytomas, and parathyroid hyperplasia. Patients with the rarer multiple endocrine neoplasia type 2B (MEN 2B) syndrome develop MTC and pheochromocytomas, as well as mucosal neuromas, ganglioneuromatosis of the gastrointestinal tract, and a characteristic "marfanoid" habitus. Finally, MTC is transmitted in an autosomal dominant pattern in some families without associated pheochromocytomas or parathyroid hyperplasia (familial medullary thyroid carcinoma, MTC1(2). Sixty-one members of two well-characterized kindreds segregating MTC1 and 34 [corrected] members of six families segregating MEN2B were genotyped using a panel of RFLP probes from the pericentromeric region of chromosome 10 near a locus for MEN 2A. Statistically significant linkage was observed between the chromosome 10 centromere-specific marker D10Z1 and MTC1 (maximum pairwise lod score 5.88 with 0% recombination) and D10Z1 and MEN2B (maximum pairwise lod score 3.58 with 0% recombination). A maximum multipoint lod score of 4.08 was obtained for MEN2B at the position of D10Z1. In addition, 92 members of a previously unreported large MEN2A kindred were genotyped, and linkage to the pericentromeric region of chromosome 10 is reported (maximum pairwise lod score of 11.33 with 0% recombination between MEN2A and RBP3). These results demonstrate that both a locus for familial MTC and a locus for MEN 2B map to the pericentromeric region of chromosome 10, in the same region as a locus for MEN 2A. The finding that each of these three clinically distinct familial cancer syndromes maps to the same chromosomal region suggests that all are allelic mutations at the same locus or represent a cluster of genes involved in the regulation of neuroendocrine tissue development.     

Diagnosis and surgical treatment of multiple endocrine neoplasia type 2A

Background

This study aims to introduce the diagnosis and surgical treatment of the rare disease multiple endocrine neoplasia type 2A (MEN 2A).

Methods

Thirteen cases of MEN 2A were diagnosed as medullary thyroid carcinoma (MTC) and pheochromocytoma by biochemical tests and imaging examination. They were treated by bilateral adrenal tumor excision or laparoscopic surgery.

Results

Nine patients were treated by bilateral adrenal tumor excision and the remaining four were treated by laparoscopic surgery for pheochromocytoma. Ten patients were treated by total thyroidectomy and bilateral lymph nodes dissection and the remaining three were treated by unilateral thyroidectomy for MTC. Up to now, three patients have died of MTC distant metastasis.

Conclusions

We confirmed that MEN 2A can be diagnosed by biochemical tests and imaging examination when genetic testing is not available. Surgical excision is the predominant way to treat MEN 2A; pheochromocytoma should be excised at first when pheochromocytoma and MTC occur simultaneously.     

Linked markers flanking the gene for multiple endocrine neoplasia type 2A

The inherited cancer syndrome multiple endocrine neoplasia type 2A (MEN2A) has recently been mapped to chromosome 10. We have typed 29 families with this disorder with DNA markers from the pericentromeric region of chromosome 10. Two markers, RBP3 and MCK2, were tightly linked to the MEN2A gene at recombination fractions of less than 3%. Multipoint analysis of the linkage data suggests that the gene is located within a 3-cM interval defined by the markers RBP3/MCK2 on one side and TB14.34 on the other. No evidence for locus heterogeneity was detected in any of the 27 families from 14 countries who were informative for the markers tested. The data confirm and refine the original assignment and provide the basis for presymptomatic screening for this disorder.     

Germ-line mutation analysis in patients with multiple endocrine neoplasia type 1 and related disorders.

Multiple endocrine neoplasia type 1 (MEN1) is an autosomal dominant syndrome predisposing to tumors of the parathyroid, endocrine pancreas, anterior pituitary, adrenal glands, and diffuse neuroendocrine tissues. The MEN1 gene has been assigned, by linkage analysis and loss of heterozygosity, to chromosome 11q13 and recently has been identified by positional cloning. In this study, a total of 84 families and/or isolated patients with either MEN1 or MEN1-related inherited endocrine tumors were screened for MEN1 germ-line mutations, by heteroduplex and sequence analysis of the MEN1 gene-coding region and untranslated exon 1. Germ-line MEN1 alterations were identified in 47/54 (87%) MEN1 families, in 9/11 (82%) isolated MEN1 patients, and in only 6/19 (31.5%) atypical MEN1-related inherited cases. We characterized 52 distinct mutations in a total of 62 MEN1 germ-line alterations. Thirty-five of the 52 mutations were frameshifts and nonsense mutations predicted to encode for a truncated MEN1 protein. We identified eight missense mutations and five in-frame deletions over the entire coding sequence. Six mutations were observed more than once in familial MEN1. Haplotype analysis in families with identical mutations indicate that these occurrences reflected mainly independent mutational events. No MEN1 germ-line mutations were found in 7/54 (13%) MEN1 families, in 2/11 (18%) isolated MEN1 cases, in 13/19 (68. 5%) MEN1-related cases, and in a kindred with familial isolated hyperparathyroidism. Two hundred twenty gene carriers (167 affected and 53 unaffected) were identified. No evidence of genotype-phenotype correlation was found. Age-related penetrance was estimated to be >95% at age >30 years. Our results add to the diversity of MEN1 germ-line mutations and provide new tools in genetic screening of MEN1 and clinically related cases.     

Characterization of mutations in patients with multiple endocrine neoplasia type 1.

Multiple endocrine neoplasia type 1 (MEN1) is an autosomal dominant disorder characterized by tumors of the parathyroids, pancreatic islets, and anterior pituitary. The MEN1 gene, on chromosome 11q13, has recently been cloned, and mutations have been identified. We have characterized such MEN1 mutations, assessed the reliability of SSCP analysis for the detection of these mutations, and estimated the age-related penetrance for MEN1. Sixty-three unrelated MEN1 kindreds (195 affected and 396 unaffected members) were investigated for mutations in the 2,790-bp coding region and splice sites, by SSCP and DNA sequence analysis. We identified 47 mutations (12 nonsense mutations, 21 deletions, 7 insertions, 1 donor splice-site mutation, and 6 missense mutations), that were scattered throughout the coding region, together with six polymorphisms that had heterozygosity frequencies of 2%-44%. More than 10% of the mutations arose de novo, and four mutation hot spots accounted for >25% of the mutations. SSCP was found to be a sensitive and specific mutational screening method that detected >85% of the mutations. Two hundred and one MEN1 mutant-gene carriers (155 affected and 46 unaffected) were identified, and these helped to define the age-related penetrance of MEN1 as 7%, 52%, 87%, 98%, 99%, and 100% at 10, 20, 30, 40, 50, and 60 years of age, respectively. These results provide the basis for a molecular-genetic screening approach that will supplement the clinical evaluation and genetic counseling of members of MEN1 families.     

Linkage analysis of a DNA marker localized to 20p12 and multiple endocrine neoplasia type 2A.

A DNA segment D20S5 isolated from a chromosome 19/20 flow-sorted library was shown to identify two restriction fragment length polymorphisms (RFLPs) with MspI and PvuII. The probe was localized by hybridization in situ to 20p12, the putative site of an interstitial deletion in some MEN 2A and 2B patients. Linkage of the D20S5 and MEN 2A loci was excluded at theta less than or equal to .13 using two large MEN 2A kindreds. These data suggest that the MEN 2A locus may not lie within 20p12 as previously suggested.     

Linkage analysis of multiple endocrine neoplasia type 1 with INT2 and other markers on chromosome 11

We evaluated linkage between the locus for multiple endocrine neoplasia type 1 (MEN1) and several polymorphic DNA markers on chromosome 11 in a single large pedigree. On the basis of the finding of a basic fibroblast growth factor (bFGF)-like substance circulating in plasma of MEN1 patients, we chose a bFGF-related gene known to be localized to 11q13 as one of the markers. This gene locus, INT2, was found to be closely linked to the MEN1 gene. Pairwise and multipoint analyses with INT2 confirm the recent finding by C. Larsson et al. (1988, Nature (London) 332: 85-87) of MEN1 linkage to another marker, skeletal muscle glycogen phosphorylase, at 11q13.     

A two-hit model for development of multiple endocrine neoplasia type 2B by RET mutations

Multiple endocrine neoplasia (MEN) type 2B mutations have been reported at methionine 918 or alanine 883 in the tyrosine kinase domain of the RET proto-oncogene. Recently, a new combination of two germline missense mutations at valine 804 and tyrosine 806 was identified in a patient with MEN 2B-like clinical phenotypes including medullary thyroid carcinoma, mucosal neuroma, and marfanoid habitus. In this case, valine 804 and tyrosine 806 were replaced with methionine and cysteine, respectively. In the present study, biological activities of RET with these new mutations were compared with those with known MEN 2A or MEN 2B mutations. The transforming activity of RET with the V804M/Y806C mutation was about 8- to 13-fold higher than that of RET with a single V804M or Y806C mutation. Like RET with the M918T or A883F MEN 2B mutation, the transforming activity of RET with the V804M/Y806C mutation was not affected by substitution of phenylalanine for tyrosine 905 that abolished the activity of RET with the MEN 2A mutation. On the other hand, substitution of phenylalanine for tyrosines 864 and 952 drastically diminished the activity of RET with the V804M/Y806C, M918T or A883F mutation, suggesting that these three mutant proteins have similar biological properties.     

Chromogranin a expression in phaeochromocytomas associated with von Hippel-Lindau syndrome and multiple endocrine neoplasia type 2.

Chromogranin A (CGA) is a major secretory protein present in the soluble matrix of chromaffin granules of neuroendocrine cells and tumours, such as phaeochromocytomas. CGA has several functions, some of which may be involved in the distinct phenotypic differences of phaeochromocytomas in patients with von Hippel-Lindau (VHL) syndrome compared to multiple endocrine neoplasia type 2 (MEN 2). In this study, we therefore compared tumour and plasma levels of CGA in patients with phaeochromocytoma associated with the two syndromes. We show that phaeochromocytomas from MEN 2 patients express substantially more CGA than tumours from VHL patients at both the mRNA (3-fold greater) and protein (20-fold) level. We further show that relative to increases in plasma catecholamines, patients with phaeochromocytomas associated with MEN 2 have higher plasma concentrations of CGA than those with tumours in VHL syndrome. These data supplement other observations that phaeochromocytomas in VHL compared to MEN 2 patients express lower amounts of catecholamines and other chromaffin granule cargo, such as chromogranin B and neuropeptide Y. Possibly the differences in tumour CGA expression may contribute to differences in secretory vesicle formation and secretion in the two types of tumours. Alternatively the differences in expression in CGA and other secretory constituents may reflect downregulation of the entire regulated secretory pathway in VHL compared to MEN 2 tumours.     

Exclusion of linkage of loci on chromosome 19 with multiple endocrine neoplasia, type 2

Linkage between seven loci on chromosome 19 and multiple endocrine neoplasia type 2a (MEN2A) was examined in a single large Swedish pedigree. A total of 50 cM was excluded from the male genetic map by pairwise analysis and an estimated 63 cM by multipoint analysis. Using existing data on the likelihood of different marker-marker distances and taking into account current exclusions on other chromosomes, the probability that the gene for MEN2A segregating in this pedigree could still be located on chromosome 19 is approximately 0.28%.     

Development of multiple endocrine neoplasia type 2A does not involve substantial deletions of chromosome 10

In MEN2A both familial and sporadic cases are known. The familial cases show a dominant pattern of inheritance. In these respects, MEN2A resembles other tumors in whose etiology so-called tumor suppressor genes play a decisive role. The MEN2A locus has been assigned to chromosome 10 by linkage studies. Analysis of tumor DNA from 42 patients shows that markers on chromosome 10 were lost in only one tumor. Thus, these results contrast with previous studies which show that tumor development is generally associated with the loss of the whole or substantial parts of the chromosome on which the putative tumor suppressor gene is located.     

Cortical-sparing laparoscopic adrenalectomy in a patient with multiple endocrine neoplasia type IIA

We describe the case of a patient affected by multiple endocrine neoplasia type IIA with a new diagnosis of an asymptomatic right pheochromocytoma. The patient underwent laparoscopic adrenalectomy with adrenal sparing. The removal of the tumor was successful with preservation of about one third of the adrenal gland. At the time of the last follow-up, the patient is well with partial hypoadrenalism without replacement therapy. The limitations to cortical-sparing adrenalectomy imposed by traditional open surgery (small tumor with peripheral location) can be reconsidered using the laparoscopic approach. Laparoscopic cortical-sparing adrenalectomy should become the gold standard for treatment of bilateral pheochromocytoma. The advantages of this technique are its efficacy and its reduced invasiveness with a low rate of complications either during the operation or in the postoperative period. Moreover, the preservation of a portion of the adrenal cortex may prevent the need for a life-long steroid replacement therapy.     

Germline mutations of the RET proto-oncogene in eight Japanese patients with multiple endocrine neoplasia type 2A (MEN2A)

Multiple endocrine neoplasia type 2A (MEN2A) is a dominantly inherited cancer syndrome characterized by medullary thyroid carcinoma, pheochromocytoma, and parathyroid hyperplasia. The gene responsible for MEN2A was localized by linkage analysis to chromosome 10q11.2 in 1987, and recently mutations in RET, a proto-oncogene in the candidate region, were discovered in patients with MEN. The majority of mutations found so far in MEN2A patients have been located in nucleotide sequences encoding cysteine residues in the extracellular domain of RET. To characterize MEN2A germline alterations in the Japanese population, we screened DNA from eight unrelated patients for mutations in exons 10 and 11 of the RET proto-oncogene and found mutations in all eight patients, at codons 618, 620, or 634; each of these sites encodes a cysteine residue in the extracellular domain of RET. The mutations were confirmed in other affected individuals in the respective families by digestion of polymerase chain reaction (PCR) products containing the mutated codons with restriction enzymes (RsaI, CfoI, or AluI) for which cleavage sites had been generated by the specific genetic alteration. These PCR-restriction enzyme systems will be useful for genetic diagnosis in members of families carrying these mutations.